Abstract Shiga toxin (Stx) producing E. coli (STEC) and Shigella dysenteriae are foodborne pathogens that can cause severe morbidity and mortality. STEC infections can progress to either hemorrhagic colitis (HC) or life-threatening hemolytic-uremic syndrome (HUS). HUS is the most common cause of acute renal failure in US children. Presently there are no FDA approved vaccines or therapeutics against STEC or Shigella infection. Moreover, the use of antibiotics exacerbates the disease. STEC and Shigella are classified as category B pathogens of national security and public health risk. Shiga toxin has been a uniquely challenging drug target. Small molecule inhibitors of Shiga toxin enzymatic activity with high potency have not been identified. Interaction of A1 subunits with ribosomes has not been previously examined as a potential drug target. The goal of this proposal is to fill this gap by developing novel screens to identify fragment and peptide inhibitors that disrupt activity of Stx2 by inhibiting its interaction with the ribosome. We identified P stalk as the ribosome docking site of the A1 subunits of Stx1 (Stx1A1) and Stx2 (Stx2A1) and showed that an 11-mer peptide corresponding to the conserved last 11 residues of P proteins binds to Stx2A1 and inhibits its activity. These studies established toxin- ribosome interactions as a new target for inhibitor discovery. We carried out a preliminary fragment screen and identified fragments that bind to Stx2A1 with micromolar affinity. In aim 1 we propose to develop Biacore-based primary screens to identify fragments, which bind to Stx2A1 with higher affinity. We will validate the hits using activity assays and verify binding and selectivity of the inhibitors using ribosome binding and active site mutants. Medicinal chemistry will be used to optimize the selected fragments into more potent leads based on their experimental X-ray crystal structure with Stx2. In aim 2 we will develop phage displaying multiple copies of the P protein peptide to determine if multivalent display of this peptide motif will disrupt the interaction of Stx2A1 with the ribosome. We will screen random P7 phage display library to identify novel peptides that can bind to Stx2A1 more strongly than the native P protein peptide and inhibit its activity. In aim 3 we propose to solve the cryo-EM structure of Stx2 in complex with the ribosome to identify the binding sites of the P proteins to facilitate optimization of the inhibitors. We expect that our unique assays to dissect toxin-ribosome interactions and toxin activity in combination with the medicinal chemistry and structural biology expertise will lead to the development of novel tool compounds and peptides, which can provide biochemical and mechanistic insight into STEC pathogenesis.